Lab 7: Pre-Lab

Your task in Lab 7 is to carryout your experiment regarding the rate of cellular respiration and begin to analyze your data. To prepare for Lab 7, please review this pre-lab page. Once you feel confident regarding the below topics, complete the corresponding LABridge in Blackboard.
  • Introduction/Review
  • Do you know enough?
  • What would we have dome in lab?
  • LABridge
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Cellular Respiration

CONNECTION ALERT! ​​Cellular respiration is the topic of Chapter 9 in your BIOL 120 lecture. Please review your textbook as needed for this lab. 

​Energy is the currency of life: all living organisms require energy to survive and reproduce. Metabolism is the series of reactions and processes, catalyzed by enzymes, which together maintain life. These reactions fall into two types: catabolic or anabolic. These processes are the inverse of each other and in photosynthetic organisms occur in tandem as the anabolic reactions of photosynthesis create the products that are then broken down by the catabolic reactions of cellular respiration (view figure at left).
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​There are two general classes of cellular respiration that are characterized by their relative efficiency (ATP production): anaerobic (without oxygen) and aerobic (oxygenated) respiration. We are focusing on aerobic respiration in this lab, which is a highly efficient process occurring within the mitochondria of eukaryotic organisms that have higher energy requirements for survival. In a 4 step process, oxygen and glucose are used to produce energy (ATP), H2O, and CO2,​ 
  1. Glycolysis ("splitting of sugar"): This step happens in the cytoplasm. One Glucose (C6H12O6) is broken down to 2 molecules of pyruvate. Requires 2 ATP to start, produces 4 with a 2 ATP net payoff. 
  2. Pyruvate Grooming: The pyruvate from glycolysis is shuttled into the mitochondria, where it is converted to a molecule called Acetyl CoA for further breakdown.
  3. The Citric Acid Cycle: Occurs in the mitochondrial matrix. In the presence of oxygen (O2), all the hydrogens (H2) are stripped off the Acetyl CoA, two by two, to extract the electrons for making ATP, until there are no hydrogens left - and all that is left of the sugar is CO2 - a waste product - and H2O. The Citric Acid Cycle results in the production of only ~4 ATPs, but produces a lot of NADH, which will go on to the next step.
  4. The Electron Transport Chain and Chemiosmosis ("the big ATP payoff"). Occurs in the christae of the mirochondria, the folded membranes inside. Electrons from hydrogen are carried by NADH and passed down an electron transport chain. The energy release runs pumps that creat an electrochemical gradient of H+ ions. In Chemiosmosis, a big pump called ATP Synthase uses the gradient to produce A LOT of ATP.  Results in the production of ~28-32 ATPs for every molecule of glucose. 
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Review the steps of anaerobic and aerobic respiration by clicking the links in the sidebar.
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Be sure you know and understand the generalized equation for aerobic cellular respiration.

Do you know enough about crayfish?

  -From the Kentucky Wildlife Action Plan (WAP)
Abundance. There are 640 species of  freshwater crayfish (Order Decapoda) worldwide (Crandall and Buhay 2008). More than 360 species are in the US and the southeast is known as a hotspot of crayfish diversity (Taylor et al. 2007). Kentucky is inhabited by 54 species, with some under taxonomic review and others potentially awaiting discovery. Seven species are endemic to the state of Kentucky. 

Status. Freshwater crayfish are valuable indicator species of watershed health. They act as "canaries in the coal mine" and provide early warnings of ecosystem distress when populations decline. Modification of habitats, sedimentation, and dams are serious threats to freshwater crayfishes. ​Nationally, about 48% of crayfish species are of conservation concern (ranging from Vulnerable to Endangered); over a third (37%) of the Kentucky fauna falls into this category (KSNPC, 2010). Cave species are particularly at-risk from upland activities that pollute groundwater flowing into cave systems; this includes issues with chemical spills, agricultural runoff, salt from roads, and siltation from poor land use.           
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Review the links in the sidebar: Facts on KY Crayfish and Crayfish Natural History. Also, watch the short video below to see these guys in action!
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Review the links in the sidebar: Facts on KY Crayfish and Crayfish Natural History. Also, watch the short video below to see these guys in action!
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Facts on KY Crayfish
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Crayfish Natural History

What WOULD we have done in lab?

Your task was to design and conduct an experiment that tests the affects of one variable on the rate of cellular respiration in crayfish (AKA craw-fish or crawl-dad). Like most animals, they rely on aerobic cellular respiration to meet their energy demands.  
How would we have measured the rate of cellular respiration? ...By measuring the CO2 produced as a bi-product of the metabolic process!  Had we met this week, you would place your crayfish in an aquatic environment  and allow it to "respire." After a set period of time (decided by you). Then you would have removed the crayfish and analyzed how much CO2 was produced as a result of cellular respiration.  Review the following steps to understand how we would have quantified respiration rate.
  • The water you would have placed your crayfish would be slightly basic and will appear pink! This is because it would contain a pH indicator called, phenolphthalein, which is pink in the presence of a base and turns clear when it approaches neutral (pH=7).
  • As cellular respiration is occurring inside your crayfish, inside the beaker, CO2  will be expelled into the solution as a bi-product. ​In an aqueous environment, this CO2 breaks down into carbonic acid (H2CO3) then into bicarbonate (HCO3-) and H+ ions.
  • Increased concentrations of H+ ions increase the acidity in solution and lower the pH. As the pH decreases the solution will turn from pink (basic) to clear (neutral). 
  • BUT! We would not have waited for that to happen on its own, it would take far too long! ​
  • So...after your crayfish "respired" in solution for your set period time, you would remove it. The water will still appear pink. Some respiration and CO2 production HAS occurred, but not enough to change the pH  to see a color change. So we would add more acid to the solution (H2SO4), via titration, until we see a color change from pick to clear. View Diagram.​
  • Lastly, you would have used the "amount of titrant used" to calculate the micromoles of carbonic acid (H2CO3​) produced in solution using this equation. 
  • ​Instead, these values will be provided to you in the Lab 7 Protocol.
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Review the slide show below. These are considerations you would have kept in mind when setting up your experiment. There are also some helpful slides on establishing a successful control and determining the rate of cellular respiration. 
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Click here to get to WKU's blackboard to take your LABridge for this week. Be sure your Notebook Entry from last lab is ready to submit!

Lab 7: Protocol

In today's lab you will work with your lab group to conduct your experiment and begin to analyze your data. 

Exercise I. Review your research proposal and the pre-lab and revise where necessary

Exercise II. Practice virtual titrations

Exercise III. Analyze your data
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Lab Objectives: Following today's lab, you should be able to...
  • Exercise I
  • Exercise II
  • Exercise III
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Exercise I. Review & Revise

Procedure
  1. Open the Lab 7 Notebook Guide
  2. Open your research proposal on Cellular Respiration from Lab 6.
  3. Review the pre-lab.
  4. Ask the following questions and augment/revise your proposal as necessary.
    • Hypotheses remain the same?
    • Independent, dependent and confounding variables remain the same? 
    • How long does your crayfish need to respire?
    • How many trials will you do?
    • Do you need a control?  (YES) ...depending on your experiment. What should it be?
    • What kind of statistics will you use?
    • What kind of graph might you make?
  5. Write up a step-by-step summary of how you might have conducted this experiment.
  6. Lastly, create some tye of illustration or diagram of your experimental design. You can use whatever program/software you chose. Word and PowerPoint would suffice.
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Lab 7 Notebook Guide. Click to download.
Also, remember how to to determine the volume of your crayfish (in pre-lab slide show): Pour 100-200 mL of stock solution into a 250 mL graduated cylinder. Gently place your animal into the graduated cylinder. Record its volume (rise in solution level in mL). Plan to do this for every crayfish.

Exercise II. Practice Titrations

In our experiment, we would have placed the crayfish in a slightly basic (pink) solution, due to the phenolphthalein indicator. It would have undergone cellular respiration while in the beaker and generated and released CO2 as a bi-product.
In an aqueous environment, CO2 combines with water to first create carbonic acid, which is then broken down into hydrogen ions. The addition of H+ ions results in a change in pH: the pH decreases and becomes more acidic. The introduction of CO2 from the crayfish would have lowered the pH some, but not enough in 20 minutes to make it acidic enough to turn clear. ​
Remember:
  • Basic solutions are pink with the phenolphthalein indicator
  • Acidic solutions are clear with the phenolphthalein indicator
How much "work" (cellular respiration) did the crayfish due while in solution? We can determine that through titrations. We would have added drops of an acidic titrant (sulphuric acid) until the solution reached a clear endpoint.
  • If we needed to add lots of drops, then the solution was farther from the endpoint because the crayfish did not expel much CO2 and must have had a lower rate of cellular respiration.
  • If we needed to add fewer drops, then the solution was closer to the endpoint because crayfish released alot of CO2 and must have had a higher rate of cellular respiration.
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General description of titration. Please review! Click to enlarge.
In either case we can use "the amount of titrant required" to calaculate exactly how much CO2 was released by our crayfish!
  • Titration is an important skill (and actually kinda fun) so it's a bummer we don't get to do this in lab. These activities are just meant to give you sense for what titration is like. Hopefully you'll get to use this skills later on in other classes.
Procedure
  1. ​Open the Titration Virtual Lab.
  2. Enable Adobe Flash if needed. 
  3. Open the Notebook Guide and refer to the table. You will be filling in this table as you proceed through the lab.
  4. You will complete two titrations.
  5. The steps of the lab are numbered. For each of your titrations, proceed through to step 5.
  6. On step 5, be careful you do not "over titrate" and miss the endpoint. You should use the "dropwise" button to ensure this does not occur.
  7. How do you know what "endpoint" you are looking for? ​Use this pH indicator chart for help!
  8. Once you've reached the endpoint, which you can tell from the color change, you'll need to record the "Total Volume of" either the acid or base titrant you used. That value is in mLs and is at the top left of the burette. 
  9. Once you've completed two titrations and your table is complete, answer the remaining questions about the titration we would have performed in lab in this week.
  10. Lastly, view the quick video showing a phenolphthalein acid-base titration.
  11. Answer the reaming question in your Lab Notebook Guide.
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Titration Virtual Lab. Click to enter.

Analysis

Procedure
  1. Congratulations! You have just finished a very successful day in lab. Together with all your lab mates, you collected the data available in the sidebar.
  2. The Excel workbook has 3 tabs. The first is of all the titration data. The second tab has all the crayfish by volume and the thrid tab is an "Example of Your Data" and shows an example of how you might set up your data table and calacutions.
  3. Please note: The test data in the column marked "Normal Condition" will be used by MANY of you for comparison, but not all. It depends on your set up and your question.
  4. Also, remember you will be using a t-test. You can only compare two groups of data, not three. So, for example: you can compare room temp to warm, or room temp to cold, or warm to cold, but you cannot compare room temp to warm and to cold. 
  5. Download the Excel sheet and highlight the data relevant to your experiment. Copy/paste your data onto a new worksheet (tab).
  6. Before you can move on to analysis you need to use the "titrant required" to calculate the micromoles of carbonic acid (H2CO3) produced by your crayfish. Use the formula provided in the sidebar for this calculation. It is a more direct measure than "titrant required."
  7. Note: To use the equation you needs to properly identify your variables. The two conditions you tested (ex: normal vs. cold or male vs. female) will be Ne. You need to pair it with the same control condition without crayfish, your Nr.
  8. Open the second tab (labeled Carayfish Volumes) in the Lab 7 data sheet in Excel. You'll find a list of available crayfish for "testing" and their accompanying volumetric measurements (mLs). You'll need those volumes to calculate your umols of carbonic acid (H2CO3).
  9. ​You should calculate the umols of carbonic acid (H2CO3) produced by each crayfish you tested. 
  10.  If you are using SIZE as your IV, do not use V in the denominator. 
  11. You are ready to move forward. Now, what type of statistics should we use to determine if the mean respiration rate between our 2 groups is significantly different? ...hopefully this is an easy answer. Are the mean umols carbonic acid (H2CO3) produced in your two groups the same or are they different?
  12. Use the tools in the sidebar to complete your Lab Notebook Guide. You will need to create a table in Excel, a graph in Excel, run a T-test and answer a set of questions,
Remember: Statistics solve the problem of determining if "more" or "higher" or "different" than is actually enough to be important and biological relevant.  Using the principles of probability, they help us parse what we observe from randomness (chance alone) as meaning (a real difference, or a real relationship). Statistics tell us how likely we would be to make the same observations we have made, if chance and randomness were the only drivers. If the probability is very low (<5%), we refer to these patterns as significant. ​​
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Lab 7 data in Excel. Click to download.
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Calculate the umols of carbonic acid produced by each crayfish. If you are using SIZE as your IV, do not use V in the denominator. Click to enlarge.
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Visit the stats section of our research library.
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T-test Online Calculator.
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How big can they get? Check this out...our very own Dr. Huskey!

Lab 7 BIOL 120 CONNECTIONS
Section 1.6: Doing Biology
Big Picture 1: How to Think Like a Scientist
BioSkillls 2: Reading & Making Graphs
BioSkillls 3: Interpreting Standard Error and Using Statistical Tests
BioSkillls 4: Working with Probabilities
Chapter 9: Cellular Respiration

Faculty Spotlight: Dr. Noah Ashley

We are conducting experiments to identify factors that affect the physiological process of cellular respiration in crayfish. Similarly, Dr. Noah Ashley's lab works to identify physiological, immunological, and behavioral responses to various factors, like sickness and sleep loss, in mice and birds. Specifically, they are investigating the costs and benefits of the sickness response in vertebrates, the inflammatory response in sleep-deprived mice, sleep loss in migratory birds, and the sleep-wake cycle in arctic songbirds. Dr. Ashley's lab is extremely productive! His research proposals have been funded by the NSF and the NIH. Learn more here (Lab Web Page). You just might recognize one of his current graduate students!

​​Research Key Words: physiology, ornithology, eco-immunology, genomics, sleep loss, arctic song birds, migrating birds
​Recent Publication:  Cooper, L. N., Mishra, I., Ashley, N.T. 2019. Short-Term Sleep Loss Alters Cytokine Gene Expression in Brain and Peripheral Tissues and Increases Plasma Corticosterone of Zebra Finch (Taeniopygia guttata). Physiological and Biochemical Zoology 92:80-91.
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